As practical examples utilizing an oxidative characteristic reaction of photosensitive dyes, disinfection and sterilization of service water with methylene blue are well known.
Various studies have been hitherto done on light excitation activities of photosensitive dyes such as xanthines (e.g., rose bengal, fluorescein, eosin, erythrosine, etc.), phenothiazines the representative example of which is methylene blue, porphyrins and the like. And, there is a tendency to increase in the use of photosensitive dyes in the fields of clinical drugs or diagnostics of cancer.
Among these photosensitive dyes, methylene blue has extremely strong photo-oxidation activity and is advantageous in that it absorbs light of long wavelength (670 nm) which is readily passed through a living body tissue. As reported by Foote in "Mechanisms of Photosensitized Oxidation", Jan. 29, 1968, Vol. 162, pp. 963-970, it is considered that the activities due to excitation of methylene blue by light is mainly resulted from two reactions. That is, it is considered that, in the case of Type I light excitation reaction, an excited dye directly reacts with a substrate and that, in the case of Type II light excitation reaction, firstly, an excited triplet dye reacts with molecular oxygen to produce singlet oxygen, and then a substrate is oxidized with the singlet oxygen. Which type of the reaction occurs depends on concentrations of particular dye, dissolved oxygen and substrate to be used.
Recently, among these photosensitive dyes, in particular, the use of porphyrins in treatment of cancer has been reported (Analytical Chemistry, Vol. 61, Dec. 15, 1989, pp. 1367-1375). According to this report, a porphyrin is injected into a cancer tissue or in the vicinity thereof and light energy is provided from outside of the body to excite the porphyrins. Whereby singlet oxygen is produced which causes a lethal effect on cancer cells. One of the important characteristics of these dyes is that the dyes can absorb light of long wavelength (&gt;650 nm) which can be passed through a living body tissue.
Recently, as an attempt at such a cancer treatment, an improved technique has been reported in which specificity in an antigen-antibody reaction is utilized. That is, in this technique, a photosensitive porphyrin dye is bound to an antibody against cancer, whereby the cancer cells per se which are the antigen are attacked specifically (JO 2059-585-A, EP 252683). However, it is not easy to bind most of these photosensitive dyes to an antibody protein.
Thus, if a highly reactive photosensitive dye is readily available which is easy to bind to proteins and is capable of absorbing light of long wavelength (&gt;650 nm), significant improvements are expected in this art.
Another important use of photosensitive dyes is that in the field of diagnostics. For example, the photosensitive dye as described herein can be used for labeling an antibody, hapten or nucleic acid (DNA or RNA) probe. The labeled antibody, hapten or nucleic acid probe thus obtained is used to produce a signal indicating the original amount of an analyte in the course of a clinical chemical analysis. The signal is derived from the labeled photosensitive dye and a representative example thereof is color development, fluorescence or chemiluminescence. However, this technique has scarcely been employed because it is difficult to obtain a highly reactive derivative of photosensitive dye which is capable of covalently binding to a protein, hapten or nucleic acid.
The desired derivative of a photosensitive dye is that having an active functional group which can readily react with a protein, hapten or nucleic acid under normal reaction conditions. One of these active functional groups is a succinimido ester group which can react with an amino group of proteins or nucleic acids. Another group is a maleimide group which can react with a thiol group of proteins. However, it is difficult to introduce these functional groups directly into methylene blue dye. This is clear from the fact that any methylene blue derivative capable of modifying proteins or nucleic acids is not yet found even though methylene blue has been studied for more than 50 years.
When an aromatic compound having low molecular weight such as a dye is bound to a protein, hapten (e.g., thyroid hormone) or nucleic acid, problems often arise such as sedimentation of a protein-dye conjugate and non-specific binding of the protein, hapten or nucleic acid to the surface of a solid phase. These problems are caused by hydrophobic nature of the dye in an aqueous solution, which results in a low solubility of the protein-dye conjugate and non-specific absorption of the protein, hapten or nucleic acid to the surface of the solid phase. Thus, if any technique to improve such an instability of a protein-dye conjugate or to prevent such non-specific adsorption, it is possible to improve conventional techniques to a great extent.